Download Cryptic impacts of temperature variability on amphibian immune

4204
The Journal of Experimental Biology 216, 4204-4211
© 2013. Published by The Company of Biologists Ltd
doi:10.1242/jeb.089896
RESEARCH ARTICLE
Cryptic impacts of temperature variability on amphibian immune function
Kimberly A. Terrell1,*, Richard P. Quintero2, Suzan Murray2, John D. Kleopfer3, James B. Murphy2,
Matthew J. Evans2, Bradley D. Nissen2 and Brian Gratwicke1
1
Center for Species Survival and 2Center for Animal Care Sciences, Smithsonian Conservation Biology Institute,
National Zoological Park, 3001 Connecticut Avenue NW, Washington, DC 20008, USA and 3Virginia Department of Game and
Inland Fisheries, 3801 John Tyler Highway, Charles City, VA 23030, USA
*Author for correspondence ([email protected])
SUMMARY
Ectothermic species living in temperate regions can experience rapid and potentially stressful changes in body temperature
driven by abrupt weather changes. Yet, among amphibians, the physiological impacts of short-term temperature variation are
largely unknown. Using an ex situ population of Cryptobranchus alleganiensis, an aquatic North American salamander, we tested
the hypothesis that naturally occurring periods of temperature variation negatively impact amphibian health, either through direct
effects on immune function or by increasing physiological stress. We exposed captive salamanders to repeated cycles of
temperature fluctuations recorded in the population’s natal stream and evaluated behavioral and physiological responses,
including plasma complement activity (i.e. bacteria killing) against Pseudomonas aeruginosa, Escherichia coli and Aeromonas
hydrophila. The best-fit model (ΔAICc=0, wi=0.9992) revealed 70% greater P. aeruginosa killing after exposure to variable
temperatures and no evidence of thermal acclimation. The same model predicted 50% increased E. coli killing, but had weaker
support (ΔAICc=1.8, wi=0.2882). In contrast, plasma defenses were ineffective against A. hydrophila, and other health indicators
(leukocyte ratios, growth rates and behavioral patterns) were maintained at baseline values. Our data suggest that amphibians
can tolerate, and even benefit from, natural patterns of rapid warming/cooling. Specifically, temperature variation can elicit
increased activity of the innate immune system. This immune response may be adaptive in an unpredictable environment, and is
undetectable by conventional health indicators (and hence considered cryptic). Our findings highlight the need to consider
naturalistic patterns of temperature variation when predicting species’ susceptibility to climate change.
Key words: thermal physiology, salamander, protein complement, Cryptobranchus, climate change.
Received 19 April 2013; Accepted 6 August 2013
INTRODUCTION
Temperate-region ectotherms experience broad thermal ranges and
are therefore expected to tolerate, and in some cases benefit from,
a warmer climate (Addo-Bediako et al., 2000; Deutsch et al., 2008;
Kellermann et al., 2012; Snyder and Weathers, 1975; van Berkum,
1988). By contrast, tropical and polar ectotherms are adapted to a
narrower and more stable range of temperatures (Janzen, 1967;
Sunday et al., 2011), and are considered more vulnerable to
anthropogenic climate change (Clusella-Trullas et al., 2011; Deutsch
et al., 2008; Stillman, 2003; Tewksbury et al., 2008). While
latitudinal patterns of thermal tolerance have received less attention
among freshwater species, there is some evidence that this
relationship extends to freshwater fish (Naya and Bozinovic, 2012)
and amphibians (Duarte et al., 2012). Given that air and water
temperatures are strongly correlated in freshwater systems (Morrill
et al., 2005), it seems logical that aquatic species at low latitudes
also would be particularly susceptible to climate change. Yet none
of these predictions consider the potential for thermal variation itself
to negatively impact ectothermic physiology. These potential
impacts would be greatest in temperate regions, where climate is
relatively less stable. Although freshwater habitats are somewhat
buffered from rapid changes in air temperature, these systems can
undergo substantial warming/cooling over relatively short time
scales (see Isaak et al., 2012).
The role of short-term (i.e. daily, weekly or monthly) temperature
variation in predicting species’ responses to climate change has been
largely overlooked, but is an area of growing research interest,
particularly in amphibians (Niehaus et al., 2006; Niehaus et al., 2011;
Niehaus et al., 2012; Raffel et al., 2006; Raffel et al., 2012). Exposure
to naturalistic patterns of thermal variation accelerates maturation
and improves jumping performance in the striped marsh frog
(Limnodynastes peronii) (Niehaus et al., 2006), yet does not
influence the metabolic performance of larvae (Niehaus et al., 2011).
Rapid temperature shifts (from 25 to 15°C) increase susceptibility
to Batrachochytrium dendrobatidis in the Cuban tree frog
(Osteopilus septentrionalis), but the physiological basis of this
relationship remains unclear (Raffel et al., 2012). Intriguingly, O.
septentionalis can acclimate to a consistent (but not randomized)
series of temperature shifts, suggesting that the pattern of thermal
variation itself can influence amphibian disease susceptibility
(Raffel et al., 2012).
Although thermal acclimation is well studied among amphibians
(reviewed in Angilletta, 2009a), this research generally focuses on
acclimation to constant temperatures. Given that most ectotherms
live in thermally dynamic habitats, there is a need to better
understand how short-term temperature variation influences both
physiology and acclimation. To our knowledge, the only previous
study of the influence of naturalistic patterns of temperature
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Immune impacts of thermal variation
40
30
Temperature (°C)
variation on amphibian immune function is a field-based
investigation (Raffel et al., 2006). These investigators correlated
short-term and seasonal weather patterns to changes in leukocyte
counts (i.e. reduced numbers of circulating lymphocytes, neutrophils
and eosinophils) in the aquatic salamander Notophthalmus
viridescens (Raffel et al., 2006). Yet the functional consequences
of these changes are unclear, particularly as corticosteroid hormones
can influence the distribution of leukocytes in blood and tissue
(Davis et al., 2008).
Understanding the environmental determinants of amphibian
physiological function is important because this taxon is
experiencing declines on an unprecedented, global scale (Hof et
al., 2011; Stuart et al., 2004; Young et al., 2004). Identifying
potential sources of immune suppression is essential (Blaustein
et al., 2012; Kiesecker, 2011), as extirpations have been linked
to pathogenic disease, most notably chytridiomycosis (Cheng et
al., 2011; Lips et al., 2006) and Ranavirus (Miller et al., 2011).
Yet for many amphibians, particularly North American
salamanders, the causes of dwindling population numbers are
enigmatic (Caruso and Lips, 2013; Highton, 2005; Stuart et al.,
2004). Climate change is hypothesized to be a contributing factor
to ongoing declines (Stuart et al., 2004) and a major driver of
future extinctions among Appalachian salamanders (Milanovich
et al., 2010). Understanding how temperature variation influences
amphibian physiology may help elucidate the causes of ongoing
declines and will be important in accurately predicting extinction
risk from climate change.
The hellbender [Cryptobranchus alleganiensis (Daudin 1803)]
is a fully aquatic salamander inhabiting rivers throughout the
Appalachian region of the eastern USA and in parts of the Ozarks
(Nickerson and Mays, 1973). Both the Ozark subspecies (C. a.
bishopi) and its eastern counterpart (C. a. alleganiensis) have
experienced widespread extirpations and population declines, likely
due in part to habitat degradation and poaching (Furniss, 2003;
Mayasich et al., 2003; Wheeler et al., 2003). Lack of recruitment
has been observed in declining populations, suggesting impaired
reproduction or increased juvenile mortality (Wheeler et al., 2003).
Intriguingly, C. alleganiensis is known to experience persistent,
idiopathic skin lesions that can progress to tissue degeneration and,
in severe cases, loss of all four feet (Nickerson et al., 2011). Although
the cause remains unknown, combinations of opportunistic
pathogens have been identified on afflicted salamanders, leading to
speculation that these individuals may suffer from environmentally
based immune suppression (Nickerson et al., 2011). A better
understanding of the relationship between thermal variation and
immune function in C. alleganiensis could perhaps provide clues
to the etiology of this enigmatic disease.
We empirically tested the physiological and behavioral impacts
of naturalistic, short-term temperature variation on C. a.
alleganiensis under controlled laboratory conditions. We measured
indicators of innate immune function, physiological stress and
overall health in juvenile salamanders before, during and after a
period of temperature changes that mimicked naturally occurring
thermal variation in the population’s natal stream (Fig. 1). Response
variables included the ability of plasma complement proteins to kill
bacterial pathogens (Escherichia coli, Pseudomonas aeruginosa and
Aeromonas hydrophila) found on diseased C. alleganiensis
(described above) (Nickerson et al., 2011), as well as proportions
of circulating leukocytes, growth rates and behavioral activity
patterns. We focused on leukocytes that are known to increase
(neutrophils and eosinophils) or decrease (lymphocytes) in
circulation when corticosteroids are elevated (Davis et al., 2008).
4205
20
10
0
0
1
2
3
4
5
6
7
Day
Fig. 1. Representative 7 day thermal cycle for Cryptobranchus alleganiensis
salamanders (N=9 per group) maintained at constant temperatures (green
line) versus variable temperatures (purple line) mimicking fluctuations in the
population’s natal stream (black line). Temperatures were within the
species’ normal thermal range (dashed blue lines) and below its critical
thermal maximum (red line). The lower limit of the thermal range (0°C) has
been offset for clarity.
Given the previously reported link between thermal variation and
amphibian disease susceptibility (Raffel et al., 2012), our hypotheses
were that (1) naturally occurring periods of relatively unstable
temperatures cause physiological stress and compromise salamander
immune function, and (2) individuals become acclimated to
temperature patterns and regain physiological function after 3 to
5 weeks of continued exposure.
MATERIALS AND METHODS
Animal husbandry
Eighteen captive-reared juvenile C. alleganiensis alleganiensis
(aged 3 years, sex unknown) were maintained in groups of three
among six independent 151 liter aquaria, each with a 114 liter sump
tank on a closed-loop pumping system. Each aquarium was equipped
with pebble substrate and natural rock hides, and independent
heating, cooling and filtration systems. Aquaria were illuminated
by fluorescent ceiling lights on an astronomic timer that was
programmed to the dates when stream temperature data were
collected (i.e. August–September 2011). Hellbenders were fed
(15 g per tank) a mixed, commercially produced diet of chopped
earthworms, live ghost shrimp and frozen/thawed crayfish or krill
twice a week. Tank water was partially (30%) changed ~1 h after
feeding using gravel siphoning to remove detritus and uneaten food
items (live shrimp remained until eaten). Tanks were replenished
with municipal water of the same temperature that had been passed
through a two-stage carbon filter. Water chemistry (ammonia, free
chlorine, total chlorine and pH) was tested once a week in a randomly
selected tank using commercially available kits. Ammonia ranged
from 0 to 0.04 ppm (mean ± s.d.=0.014±0.012 ppm) and total
chlorine ranged from 0 to 0.01 ppm (0.002±0.004 ppm). Free
chlorine was not detected. Water pH ranged from 7.5 to 7.8
(7.76±0.10) and was within the range of values recorded in
hellbender streams during recent field surveys (6.52–8.36).
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4206 The Journal of Experimental Biology 216 (22)
Table 1. Magnitude of thermal variation of C. alleganiensis stream
habitat in southwestern New York from August 2011 to August
2012
provide power to either the heater or the chiller, which we partially
compensated for by allowing temperatures of the control group (N=9,
maintained at 21°C) to fluctuate by ±1°C (Fig. 1).
Absolute temperature change (°C)
Time scale
Hourly
Daily
Weekly
Monthly
Minimum
Mean ± s.d.
Maximum
0
<0.1
0.9
4.0
0.14±0.16
1.9±1.3
5.3±2.3
9.5±3.3
3.9
7.4
11.1
17.7
Temperature experiment
Water temperatures were recorded every 30 min over 12 months using
a HOBO Water Temperature Pro v2 Data Logger (U22-001, Onset
Computer Corporation, Bourne, MA, USA) that was secured on the
stream bed, adjacent to the ‘nest rock’ in southern New York where
the hellbenders were obtained as embryos. Temperatures ranged from
0 to 29°C, and varied significantly on hourly, daily, weekly and
monthly time scales (Table 1). Most thermal variation occurred during
the spring and summer, whereas winter temperatures were stable
across all time scales (Table 2). We identified five periods where rapid
warming/cooling occurred (Δ10–11°C in 7 days) that began in midMarch, late April, late May, late August and late September. We
selected a 7 day period (30 August–5 September 2011) where
temperatures ranged from 16 to 26°C, because we could maintain the
laboratory within this range and thus minimize changes in body
temperature during blood collection. Although there is little historical
stream temperature data for this region (as is the case in most regions),
we consider these temperatures to be within the population’s normal
thermal range. Mean air temperatures in this region were higher than
normal during August and September 2011, but still lower than
average July temperatures (National Climatic Data Center, 2013).
We reproduced the above pattern of temperature variation at an
hourly scale (Fig. 1) using a custom-engineered system and Siemens
Insight Workstation (Siemens Corporation, Washington, DC, USA).
We exposed captive hellbenders (N=9) to six consecutive 7 day
temperature cycles (42 days total). This number of cycles allowed
us to test for evidence of thermal acclimation through longitudinal
sampling, while providing individuals with 14 days to recover from
each blood collection. Physiological data collection, feeding and
water changes were carried out at times in the temperature cycle
when all aquaria were at 21°C. The laboratory was maintained at
21°C to minimize changes in hellbender body temperature during
physiological assessments. Overall, our automated system was
highly effective at recreating the 7 day pattern of warming/cooling,
with our experimental temperatures differing from corresponding
in situ values by a mean of 0.3±1.9°C (Fig. 1). This modest
variation was likely due to the system’s requirement to continually
Table 2. Seasonal patterns of thermal variation of C. alleganiensis
stream habitat in southwestern New York from August 2011 to
August 2012
Seasons representing the mosta:
Time scale
Hourly
Daily
Weekly
Monthly
Variable temperatures
Stable temperatures
Summer
Summer
Spring, Fall
Spring
Year-round
Winter, Fall, Spring
Winter
Winter, Fall
Spring: March–May; Summer: June–August; Fall: September–November;
Winter: December–February.
a
Represented within the corresponding 90th percentiles.
Blood collection
We collected blood from each salamander every 2 weeks during the
experimental period, i.e. 1, 3 and 5 weeks after the onset of temperature
fluctuations. To determine baseline values, we also collected blood
6 and 3 weeks before the experimental period, and 1 and 3 weeks
after the experimental period. Blood (≤0.4 ml) was collected from the
caudal tail vein using a 22 gauge needle and a 1 ml syringe within
4 min of capture. Each animal was weighed in a separate sterile
container after blood draw. Towels and gloves were changed between
successive animals. A small volume (5 μl) of blood was immediately
smeared onto a glass microscope slide in duplicate for leukocyte
counts. After drying, the slides were fixed in 100% methanol for 5 min,
stained with DipQuick (MWI Veterinary Supply, Boise, ID, USA),
and examined at 1000× magnification using a standard light
microscope. For each smear, 100 leukocytes were counted and
identified as neutrophils, lymphocytes, eosinophils or monocytes
(Heatley and Johnson, 2009). Proportions of each cell type were
averaged between duplicate smears. The remainder of each blood
sample was transferred to a heparinized tube and centrifuged (15 min,
3200 g) within 2 h to isolate plasma for bacterial killing assays. Plasma
samples were frozen at −80°C until analysis.
Bacteria-killing assay
Bacteria-killing ability of C. alleganiensis plasma was tested
separately against E. coli (ATCC8739), P. aeruginosa (ATCC 9027)
and A. hydrophila (ATCC 35654) using a modification of the
absorbance-based protocol described previously (Liebl and Martin,
2009). Bacteria were obtained as lyophilized pellets (Fisher Scientific,
Waltham, MA, USA) and rehydrated in 10 ml amphibian phosphate
buffered saline (PBS). An isolated colony of each bacterium was
obtained by streaking onto a 5% blood agar plate (Fisher Scientific)
and used to inoculate 10 ml of tryptic soy broth. The broth was
incubated for 24 h at 30°C to yield a working stock solution. Bacterial
concentration of each stock solution was determined by plating serial
dilutions (10−4, 10−5, 10−6, 10−7) onto 5% blood agar. Plates containing
30–300 isolated colonies were counted to determine colony-forming
units (CFUs) per milliliter. For each sample, 5 μl of thawed plasma
was combined with 20 μl of bacteria (diluted to 106 CFUs ml–1) and
75 μl of amphibian PBS in duplicate in a 96-well plate. A blank
(plasma and PBS only) was included for each sample. Positive
(plasma-free) and negative (bacteria-free) controls were included in
each plate in triplicate. Plates were shaken and subsequently incubated
(21°C, 1 h) to allow bacteria killing to occur. Tryptic soy broth (200 μl)
was then added to each well (except for blanks), and plates were
incubated (30°C) for 10 h (A. hydrophila), 12 h (E. coli) or 16 h (P.
aeruginosa) to allow bacterial growth to occur. Length of incubation
corresponded to the amount of time needed for the bacterium to reach
its exponential growth phase, which we previously determined by
measuring changes in sample absorbance over time. Absorbance was
read at 405 nm immediately after incubation. Bacteria-killing ability
was calculated as the difference in absorbance between the sample
and the blank, divided by the positive control.
Behavioral observations
We observed diurnal (11:00–14:00 h) and feeding (14:00–16:00 h)
behaviors once a week throughout the experimental period. Nocturnal
(22:00–02:00 h) behaviors were observed twice a week during the
same period. For each tank, salamander behavioral state (active versus
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Immune impacts of thermal variation
4207
Table 3. Linear mixed effects models of physiological responses to temperature change
Model
Fixed effects
Date + Study period + Treatment groupa
Date + (Study period × Treatment group)
Date + (Offset study period × Treatment group)b
(Date × Study period) + (Study period × Treatment group)
(Date × Offset study period) + (Offset study period × Treatment group)
Null
Rapid and sustained response
Delayed and sustained response
Rapid response followed by acclimation
Delayed response followed by acclimation
Individual was included as a random effect in each model.
a
Treatment effect was represented by an interaction between treatment group and study period, since there could be no effect of temperature change during
baseline data collection.
b
Study period was offset by 2 weeks to represent a delayed treatment effect.
inactive) was recorded as a point observation every 30 s over 3 min.
Active behaviors included swimming, crawling and floating. Animals
that were stationary and in contact with the gravel substrate were
considered inactive. Side-to-side rocking (a potential sign of stress)
was recorded as a behavioral event if it was observed between point
observations. Each 3 min observation period was repeated twice, for
a total of 9 min per tank. Because individuals could not be reliably
identified during observations, we recorded the total number of
salamanders in the tank engaging in each behavior. This number was
summed and averaged among replicate sets of observations.
We analyzed behavioral data in a hypothesis-testing framework
because observations were made during the experimental period only
(and hence we directly compared treatment and control groups).
We tested for an effect of temperature fluctuations using a linear
mixed model with treatment group included as a fixed effect. Tank
was included as a random effect because individuals housed together
frequently interacted with one another (e.g. through direct contact),
likely influencing behavioral activity. Results were considered
significant at P<0.05.
Statistical analyses
Comparison of linear mixed effects (LME) models based on weights
calculated from AICc values revealed a single supported model for
all physiological response variables except E. coli killing (Table 4).
We found strong support (ΔAICc=0, wi>0.99) for an effect of
temperature variation on P. aeruginosa killing. This model included
a 1–3 week delay in physiological response, but no thermal
acclimation effect, and predicted a 70% increase in killing ability
after exposure to variable temperatures (Fig. 2A). The same model
predicted a 53% increase in E. coli killing (Fig. 2B) but had less
support (ΔAICc=1.8, wi=0.29). Escherichia coli killing data better
supported the null model, which represented no effect of temperature
variation (ΔAICc=0, wi=0.71). Paired t-test comparison of all
control group samples (N=53) revealed that plasma proteins were
more effective (P<0.0001) at killing E. coli compared with P.
aeruginosa, with a mean of 56±39% and 37±14% of bacterial cells
killed, respectively. In contrast, plasma proteins were completely
ineffective against A. hydrophila (i.e. 0% of bacterial cells were
killed in every sample).
Across all samples (N=122), lymphocytes were by far the
predominant leukocyte (mean ± s.d., 94.7±3.5%), with lower
percentages of neutrophils (2.5±2.6%) and eosinophils (1.0±1.1%)
present in circulation (Fig. 3). Monocytes also were observed
infrequently (1.8±1.7%) and basophils were not present. Model
comparisons (Table 4) revealed strong support for the null model
RESULTS
All data were analyzed using R statistical software (R Development
Core Team, 2008). We compared linear mixed models within each
response variable using the lmer function in the lme4 package (Bates
et al., 2012) and calculated Akaike weights (wi) for each model from
corrected Akaike’s information criterion (AICc) values. We used an
AICc model comparison approach in order to investigate the potential
for a delayed response to temperature changes as well as an
acclimation effect. Model terms are provided in Table 3. A rapid
response assumed that physiology was altered within 1 week of the
onset/cessation of temperature fluctuations (i.e. before the subsequent
blood collection). A delayed response assumed that this impact
occurred between 1 and 3 weeks (i.e. after the subsequent blood
collection). To compare all models using a single data set, study period
was included as two distinct predictor variables, representing either
a rapid or a delayed response. Tank was included in the initial analysis
as a random effect, but did not influence AIC values of any treatment
model. Because few tanks (N=3) were nested within each treatment,
we omitted tank from the final analysis to avoid representing a
treatment effect in the null model. We plotted estimates and standard
errors for treatment effects models that had an Akaike weight (wi) of
>0.01. If there was no support for an effect of temperature treatment
on a given response variable (i.e. wi≤0.01), we plotted estimates and
standard errors of the null model.
Table 4. Akaike weights (wi) for linear mixed effects models of physiological responses to temperature change
Model
a
Response variable
Null
Rapid and sustained
Delayed and sustained
Acclimationb
P. aeruginosa killing
E. coli killing
Lymphocytes
Neutrophils
Eosinophils
Growth rates
0
0.71
>0.99
>0.99
>0.99
>0.99
0
0
<0.01
<0.01
<0.01
<0.01
>0.99
0.29
<0.01
<0.01
<0.01
<0.01
<0.01
0
0
0
0
0
a
No effect of temperature fluctuations.
Includes a delayed response term. No support (wi=0) was observed for the acclimation model with a rapid response term.
Bold values indicate strong support (wi>0.99) for the corresponding mixed effects model.
b
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4208 The Journal of Experimental Biology 216 (22)
A
120
B
100
60
●
45
●
●
30
●
E. coli killed (%)
P. aeruginosa killed (%)
75
15
●
80
60
●
●
●
40
20
0
Baseline
0
Experimental
Study period
Baseline
(i.e. no effect of temperature fluctuations) in leukocyte data
(ΔAICc=0, wi>0.99; Fig. 3) and growth rates (ΔAICc=0, wi>0.99;
Fig. 4).
As expected, salamanders were more active (P<0.0001) during
nocturnal versus diurnal hours and most active (P=0.002) during
feeding periods (Fig. 5A). Temperature fluctuations did not influence
the frequency (P=0.355) or temporal pattern (P=0.319) of active
behaviors. Rocking behavior was observed infrequently (<5% of
observations) and was not influenced by temperature fluctuations
(P=0.207; Fig. 5B).
DISCUSSION
Current understanding of amphibian thermal physiology is based
largely on the influence of absolute temperatures, especially the
limits of heat/cold tolerance (Angilletta, 2009b). To our knowledge,
this was the first study to empirically test the influence of
temperature variation on amphibian immune function by mimicking
natural, fine-scale patterns of warming/cooling. Our study yielded
four major findings. First, and contrary to our expectations, variable
100
Cell count (% of circulating leukocytes)
Fig. 2. Linear mixed model estimates and standard
errors for (A) Pseudomonas aeruginosa (ΔAICc=0,
wi>0.99) and (B) Escherichia coli (ΔAICc=1.8,
wi=0.29) killing ability in Cryptobranchus
alleganiensis salamanders exposed to variable
temperatures (N=9, solid error bars) during the
experimental period compared with a control group
maintained at constant temperatures (N=9, dashed
error bars). Model terms included a 1–3 week
delayed treatment response but no acclimation
effect.
80
60
40
20
0
Baseline
Experimental
Study period
Fig. 3. Estimates and standard errors for the null model (ΔAICc=0, wi>0.99) of
circulating leukocyte ratios in Cryptobranchus alleganiensis salamanders
exposed to variable temperatures (N=9, solid error bars) during the
experimental period compared with a control group maintained at constant
temperatures (N=9, dashed error bars). Leukocytes include lymphocytes (red
markers), neutrophils (green markers) and eosinophils (blue markers), and
representative images of each cell type in C. alleganiensis are illustrated.
Experimental
temperatures enhanced (rather than suppressed) innate immune
function in salamanders, as demonstrated by the increased ability
of plasma proteins to kill P. aeruginosa. Because the P. aeruginosa
bacterium has been isolated from open sores on wild C. alleganiensis
(Nickerson et al., 2011) and is a common cause of disease among
vertebrate taxa, this response likely translates to a direct increase
in fitness. Second, increased immune function was sustained over
the 6 week experimental period, indicating that salamanders did not
acclimate to this complex pattern of temperature change. Third,
salamanders maintained normal (i.e. baseline) levels of circulating
immune cells, growth and behavioral activity during exposure to
rapid warming and cooling. This finding, combined with the
observed increase in bacteria killing, suggests that natural patterns
of temperature variation benefit amphibian health. Finally, we
observed that the antimicrobial activity of C. alleganiensis plasma
varies dramatically among species of gram-negative bacteria,
highlighting the value of our comparative approach (Nickerson et
al., 2011).
Among amphibians, there is compelling evidence linking absolute
temperatures to changes in immune function. For example, continued
exposure to 5°C reduces T-lymphocyte proliferative ability and
complement activity in Rana pipiens (Green and Cohen, 1977;
Maniero and Carey, 1997) and inhibits antimicrobial peptide
synthesis in R. sylvatica (Matutte et al., 2000). Conversely, warm
(and constant) temperatures increase resistance of Ambystoma
tigrinum larvae to Ranavirus (Rojas et al., 2005) and accelerate
allograft rejection in R. esculenta, Bombina bombina, Bufo bufo and
Diemictylus viridescens (Cohen, 1966; Jozkowicz and Plytycz,
1998). Yet most natural habitats do not maintain constant
temperatures. Given that a modest temperature change [e.g. 3°C
(Cohen, 1966)] can have a significant impact on amphibian immune
function, it seems logical that continuously fluctuating temperatures
would have important consequences for disease resistance. Variable
temperatures might influence amphibian immune function by: (1)
triggering a thermal stress response, (2) directly affecting immune
cell or protein activity, or (3) allowing a pathogen to acclimate to
the thermal environment more quickly than its amphibian host [i.e.
the ‘lag’ hypothesis (Raffel et al., 2006)]. We tested the first two
mechanisms, and our data supported the second possibility, as we
documented an increase in protein complement activity without
corresponding changes in leukocyte ratios. This response could have
been driven by increased production of one or more key complement
proteins (e.g. C3) and/or changes in the expression of regulatory
proteins (e.g. decay-accelerating factor) (Janeway et al., 2001). Our
data also revealed a latency of 1–3 weeks between the onset or
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Weekly growth (% body mass increase)
Immune impacts of thermal variation
2.0
1.6
●
1.2
0.8
●
●
●
0.4
0
Baseline
Experimental
Study period
Fig. 4. Estimates and standard errors for the null model (ΔAICc=0, wi>0.99)
of growth rates in Cryptobranchus alleganiensis salamanders exposed to
variable temperatures (N=9, solid error bars) during the experimental
period compared with a control group maintained at constant temperatures
(N=9, dashed error bars).
cessation of temperature fluctuations and a corresponding change
in complement activity. This delay may reflect the time needed to
cross a physiological response threshold and/or the time required
for protein synthesis/degradation.
Although we observed elevated immune function, we found no
evidence of an acclimation response in salamanders exposed to
6 weeks of temperature fluctuations. This contrasts with recent
findings that Cuban tree frogs (O. septentrionalis) acclimate to daily
temperature shifts (25°C↔15°C) within 4 weeks of continued
exposure (Raffel et al., 2012). However, these frogs were exposed
to immediate 10°C temperature changes, whereas the salamanders
in our study experienced fine-scale, naturalistic patterns of warming
and cooling with temperatures programmed at 30 min intervals.
Taken together, these findings raise the question of whether the
complexity of a temperature pattern can influence an amphibian’s
thermal acclimation response. This question may be particularly
important for temperate-region ectotherms that experience relatively
unstable temperatures compared with tropical counterparts. In the
present study, we observed periods of substantial stream temperature
variation over hourly, daily, weekly and monthly time scales
(Table 1), particularly during the spring and summer (Table 2). In
A
10
contrast to simple temperature shifts, amphibians may be unable to
acclimate to more complex, naturalistic patterns of thermal variation
because these changes are relatively harder to predict.
Eco-physiological studies often focus on markers of biological
stress as indices of individual or population-level fitness. Yet
corticosteroid hormones are ambiguous indicators of health, and
even chronic stress may be adaptive (Boonstra, 2013). Changes in
the proportions of circulating leukocytes (specifically, more
neutrophils and fewer lymphocytes) are also widely used indicators
of physiological stress. However, this response is also ambiguous
and is believed to result from the redistribution of cells in blood
versus tissue, rather than a change in cellular production (Davis et
al., 2008). Furthermore, these and other conventional indicators of
health (e.g. growth or behavior) are less likely to reflect a positive
effect of environmental change, particularly for laboratory animals
maintained under ideal conditions. That is, any departure from
normal or baseline values would likely reflect a negative effect on
stress, growth or behavior. Yet the value of these indicators is that
they are relatively easy to quantify and can be assessed in parallel
with more direct measures of fitness to generate a better
understanding of physiological function. The consistency of
leukocyte counts, growth rates and behavioral data in the present
study, combined with an increase in innate immune function,
provides compelling evidence that amphibians can tolerate, and
perhaps benefit from, natural patterns of temperature variation.
To our knowledge, this study represents the first published
information on circulating leukocyte ratios in juvenile C.
alleganiensis. We were surprised by the predominance of
lymphocytes (>80% of immune cells in every sample), as well as
the consistency of leukocyte ratios among individuals and over time.
These findings contrast with the more variable proportions of
circulating lymphocytes (~35–65%), neutrophils (~20–45%) and
eosinophils (~3–20%) previously reported in C. alleganiensis
(Huang et al., 2010; Jerrett and Mays, 1973; Solís et al., 2007).
Because previous studies have focused on wild-caught adults, these
differences could be related to environmental variables or animal
age. Regardless, our findings highlight the need to better understand
both developmental immunology and eco-physiology in this species.
The final insight from our study was that bacteria-killing ability
of C. alleganiensis plasma differed substantially against different
pathogens. Although E. coli is a valuable model species and the
pathogen most commonly used in bacterial-killing assays, it was
also the easiest to defend against. Similar findings were reported in
a recent study of complement activity in little brown bats (Myotis
B
Fig. 5. Means and standard
errors for frequency of active
behaviors (A) and rocking (B) in
Cryptobranchus alleganiensis
salamanders exposed to
variable temperatures (N=9,
solid error bars) during the
experimental period compared
with a control group maintained
at constant temperatures (N=9,
dashed error bars). There were
no differences between
treatment groups (P≥0.207).
8
30
● ●
20
Frequency of rocking
(% of observations)
Frequency of active behaviors
(% of observations)
40
● ●
10
0
● ●
Diurnal
6
4
● ●
2
● ●
● ●
0
Nocturnal
Feeding
4209
Diurnal
Nocturnal
Feeding
Observation period
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4210 The Journal of Experimental Biology 216 (22)
lucifugus) in which a greater proportion of E. coli was killed relative
to Stapholococcus aureus and Candida albicans (Moore et al., 2011).
Thus, E. coli may represent an ‘easy test’ for the innate immune
system, allowing deficient individuals to achieve a high score. This
idea could explain why our statistical model provided relatively weak
evidence for an influence of temperature variation on E. coli killing,
despite robust support for an effect on P. aeruginosa. In contrast
to these two bacterial species, C. alleganiensis plasma was
completely ineffective against A. hydrophila. This lack of plasmabased immune defense may contribute to the high virulence of this
pathogen in wild amphibian populations, where it causes red-leg
disease (Bradford, 1991; Nyman, 1986). Alternately, a different
component of the amphibian immune system (e.g. neutrophils or
the classical complement pathway) may play a crucial role in A.
hydrophila resistance. Regardless, the observed differences among
bacterial species illustrate the value of comparative physiology and
non-traditional model species in understanding environmental
drivers of animal health.
The need for a physiologically based, mechanistic understanding
of the impacts of environmental change in species conservation is
becoming increasingly recognized (Seebacher and Franklin, 2012;
Stevenson et al., 2005; Wikelski and Cooke, 2006), and the field of
vertebrate eco-physiology has grown rapidly in the past decade (e.g.
Blaustein et al., 2012; Cooke et al., 2012; Klaassen et al., 2012;
Raubenheimer et al., 2012). To accurately predict the physiological
impacts of temperature variation on amphibians in situ, we need a
better understanding of the distinct effects of variable weather patterns
versus changes in absolute temperatures. Like dozens of Appalachian
species, C. alleganiensis is adapted to relatively cool temperatures
and will likely be influenced by a warmer climate (Milanovich et
al., 2010). Annual temperatures are predicted to increase between
~3°C (low emissions B1 scenario) and ~5°C (high emissions A1fi
scenario) throughout the Appalachian region by 2099 (Karl et al.,
2009). Identifying and predicting climate change impacts on
freshwater systems is difficult because of the lack of historical data
in these habitats (Arismendi et al., 2012), but stream temperatures
are strongly influenced by climate and typically warm by 0.6–0.8°C
for every 1°C increase in air temperature (Morrill et al., 2005; Webb
and Nobilis, 2007). Evidence of long-term warming and its biological
impacts have been observed for streams in many of the United States
(Isaak et al., 2010; Isaak et al., 2012; Kaushal et al., 2010), and models
predict that this trend will continue, both within the USA (Ruesch
et al., 2012; Wenger et al., 2011) and globally (Punzet et al., 2012).
Warmer temperatures may increase disease susceptibility and
compromise overall physiological function for species that are
pushed beyond their thermal optimum (Huey et al., 2012). This threat
is particularly acute for stream-dwelling species whose movements
are constrained within linear networks (Fagan, 2002). Yet while the
impacts of absolute temperatures are relatively well studied and can
be (to some extent) inferred from a species’ current range (Angilletta,
2009b), the effects of temperature variation itself are less obvious.
Our present findings suggest that salamander immune function
(specifically, complement activity) might be maintained, or perhaps
even enhanced, if warm periods were punctuated by cooling events.
Importantly, our data also reveal that temperature effects on
amphibian health may be cryptic (i.e. undetected by conventional
measures), underscoring the importance of non-traditional and more
direct fitness indicators. Collectively, our findings illustrate the value
of a comprehensive and ecologically relevant approach to
investigating thermal physiology. A better understanding of
amphibian thermal physiology is crucial to sustaining this remarkably
diverse taxon in the face of a rapidly changing climate.
ACKNOWLEDGEMENTS
The authors thank Ken Roblee (NY Department of Environmental Conservation)
and Penny Felski (Buffalo Zoo) for access to the research animals, and Keith
Turner (Siemens Engineering Inc.) for designing and providing the programmable
thermostat system. Kerry Grisewood provided access to her property and
valuable assistance in stream temperature data collection. We also thank our
colleagues from Smithsonian Conservation Biology Institute: Dr Janine Brown
(interpretation of results), Veronica Acosta (salamander blood collections), Dr Jeff
Hostetler (assistance with statistical analyses), Ed Bronikowski (research space
and resources), Lauren Augustine (salamander husbandry) and Emma Johnson
(data entry). Finally, we are grateful to two anonymous reviewers whose feedback
helped us significantly improve this manuscript.
AUTHOR CONTRIBUTIONS
K.A.T. designed and performed research, analyzed and interpreted data, and
wrote the paper. R.P.Q. designed and fabricated the aquatic system and
performed research. S.M. designed research and performed veterinary
procedures. J.D.K. designed research and interpreted data. J.B.M. designed
research and provided laboratory space. M.J.E. designed and performed
research. B.D.N. performed research. B.G. designed research. All authors
improved the paper.
COMPETING INTERESTS
No competing interests declared.
FUNDING
This research was supported by a David H. Smith Fellowship (to K.A.T.), which is
funded by the Cedar Tree Foundation and administered by the Society for
Conservation Biology.
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